Download The Euprymna scolopes -Vibrio fischeri Symbiosis: A Biomedical

J. Molec. Microbiol. Biotechnol. (1999) 1(1): 13-21.
The Squid-Vibrio Symbiosis
as a Biomedical
Model 13
JMMB
Symposium
The Euprymna scolopes -Vibrio fischeri
Symbiosis: A Biomedical Model for the Study
of Bacterial Colonization of Animal Tissue
Edward G. Ruby*
Pacific Biomedical Research Center
University of Hawaii, Manoa, Hawaii
Abstract
The diversity of microorganisms found in the marine
environment reflects the immense size, range of
physical conditions and energy sources, and
evolutionary age of the sea. Because associations with
living animal tissue are an important and ancient part
of the ecology of many microorganisms, it is not
surprising that the study of marine symbioses
(including both cooperative and pathogenic
interactions) has produced numerous discoveries of
biotechnological and biomedical significance. The
association between the bioluminescent bacterium
Vibrio fischeri and the sepiolid squid Euprymna
scolopes has emerged as a productive model system
for the investigation of the mechanisms by which
cooperative bacteria initiate colonization of specific
host tissues. The results of the last decade of research
on this system have begun to reveal surprising
similarities between this association and the
pathogenic associations of disease-causing Vibrio
species, including those of interest to human health
and aquaculture. Studies of the biochemical and
molecular events underlying the development of the
squid-vibrio symbiosis can be expected to continue
to increase our understanding of the factors controlling
both benign and pathogenic bacterial associations.
Introduction
Two areas of biomedical significance have emerged during
the past 20 years. The first of these is the rise in coastal
areas across the world of bacterial diseases borne by
marine and brackish-water bacteria in the genus Vibrio
(Blake, 1983; Colwell, 1996). The second is the growing
awareness of the importance of cooperative bacterial
colonization of animal tissues to the proper development
and health of the host (McFall-Ngai, 1998a; Rook and
Stanford, 1998). Progress in addressing questions in both
of these areas has been made through the investigation of
a symbiotic association involving Euprymna scolopes, a
shallow-water squid, and Vibrio fischeri, a bioluminescence
bacterium. This association has developed into a paradigm
not only for the study of symbiosis in the marine environment, but for the myriad of both cooperative and pathogenic
bacteria-animal host interactions that characterize the
*For correspondence. Email [email protected];
Tel. (808) 539-7309; Fax. (808) 599-4817.
© 1999 Horizon Scientific Press
biological world.
The principal interactions between bacteria and animal
tissue occur along the apical surfaces of host epithelial
cells. Typically, a diverse consortium of microorganisms
and host cells forms a dynamic, cooperating unit that helps
maintain a healthy microenvironment within the colonized
tissues. Such associations are ubiquitous, exhibit species
specificity and, with few exceptions (Breznak and Brune,
1994; Whittaker et al., 1996; Hooper et al., 1998), are poorly
understood. A better understanding of the process by which
bacteria and host cells exchange the signals that underlie
these phenomena is an important research goal of
biomedical research. Over the past decade the E. scolopesV. fischeri association, a natural and experimentally facile
model system, has been used to discover the nature of
the mechanisms by which animals form beneficial
associations with microorganisms, and to determine how
these mechanisms are related to, but have diverged from,
those that function in pathogenic infections. Initial studies
concentrated on a description of the system, leading to an
understanding of the light organ morphology in both the
juvenile (Montgomery and McFall-Ngai, 1993; Weis et al.,
1993; Hanlon, et al., 1997) and the adult (McFall-Ngai and
Montgomery, 1990; Montgomery and McFall-Ngai, 1998)
animal. Similarly, the pattern of growth of V. fischeri cells
during the initiation of the association (Ruby and Asato,
1993), and the role of the bacteria in the induction of its
subsequent development (Foster and McFall-Ngai, 1998;
McFall-Ngai and Ruby, 1998) were described. In addition,
unexpected phenomena were discovered, such as the
pronounced and dynamic diel rhythm dominating the state
of the association (Lee and Ruby, 1994a; Boettcher et al.,
1996), and the presence of the antibacterial enzyme
haloperoxidase within the contents of the light organ crypts
(Weis et al., 1993).
This review describes the colonization of the squid light
organ by V. fischeri , emphasizing those bacterial
determinants that have homologs or analogs among
pathogenic Vibrio species. What we have learned about
the development and specificity of this process by an
examination of the host-imposed obstacles or barriers to
colonization is also outlined. The review concludes with a
discussion of the possible roles current and future studies
of the E. scolopes-V. fischeri association may play in
biomedically important areas of research.
The Symbiotic Partners
Euprymna scolopes is a small, nocturnal, sepiolid squid
(Berry, 1912) that is bioluminescent owing to the presence
of bacterial symbionts contained within a complex, bilobed,
light-emitting organ (Nesis, 1987; McFall-Ngai and
Montgomery, 1990). Light generated by these bacteria
allows the host squid to produce a controlled, diffuse,
ventral glow as an antipredatory behavior (McFall-Ngai,
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14 Ruby
e
}
lo
*
m
g
1990). The organ lies within the center of the squid’s mantle
cavity, where it is continually bathed with ambient seawater
(Figure 1). Histological and ultrastructural analyses of the
light organ have revealed an integrated set of tissues that
serves both to support nutritionally a culture of a specific
luminous bacterium, and to control and direct the
bioluminescence they produce (Montgomery and McFallNgai, 1992; McFall-Ngai, 1994). The bacterial symbionts
are housed extracellularly within epithelium-lined spaces,
or crypts. The polarized epithelium bears a dense layer of
complex, branched microvilli within which the symbionts
are invested (Lamarcq and McFall-Ngai, 1998).
V. fischeri, the only bacterium found to naturally persist
in the light organ of E. scolopes (McFall-Ngai and Ruby,
1991), is a common marine microbe that can be easily
isolated either from this symbiosis (Boettcher and Ruby,
1990) or from light organ associations of other squids
(Fidopiastis et al., 1998) or fishes (Nealson and Hastings,
1991), and from free-living populations in the environment
(Ruby and Lee, 1998), and that can be readily grown on
both complex and minimal media. An extensive literature
exists concerning both the physiology and ecology of V.
fischeri (Nealson and Hastings, 1991), and the biochemistry
and molecular biology of its bioluminescence capability
Figure 1. The Euprymna scolopes Symbiotic Light
Organ
A ventral dissection of an anesthetized adult animal
reveals the bilobed light organ (lo) in the center of
the mantle cavity (McFall-Ngai and Montgomery,
1990), and partially occluded by the ink sac (black).
Each lateral face of the adult light organ has a single
pore through which the bacteria-containing crypts,
deep in the center of the lobes of the organ (*), are
connected to the outside environment (e, eye; m,
mantle; g, gill; bar = 1 cm).
(Meighen, 1991; Dunlap, review in this issue).
Since 1996, new advances in the development of
genetic and biochemical tools for the study of the V. fischeriE. scolopes association have been important in facilitating
progress in understanding this system. Principal among
these bacterial genetic procedures and constructs for use
in V. fischeri are:
(i) Transfer of DNA by electroporation, using the ability
to overcome innate restriction barriers in V. fischeri
(Visick and Ruby, 1997),
(ii) Chromosomal gene replacement and marker
exchange (Visick and Ruby, 1996),
(iii) Transposon mutagenesis using Tn10 (Visick and Ruby,
1997), and two mini-Tn 5 , carrying either a
chloramphenicol (Graf and Ruby, 1994) or a
streptomycin/spectinomycin (Stabb and Ruby, 1998)
resistance cassette,
(iv) Antibiotic resistance markers for erythromycin,
tetracycline, trimethoprim (from Tn7) and streptomycin/
spectinomycin (Visick and Ruby, 1997; Stabb and
Ruby, 1998),
(v) Stablely maintained and suicide plasmids (Visick and
Ruby, 1996), transferable by triparental mating (Stabb
and Ruby, 1998),
The Squid-Vibrio Symbiosis as a Biomedical Model 15
(vi) Promoter reporter constructs driving either lacZ or
luxAB genes(Visick and Ruby, 1997; 1998a),
(vii) Libraries of V. fischeri DNA restricted with EcoRI (Visick
and Ruby, 1998b), BglII or XbaI (Stabb and Ruby,
1998), and
(viii)A genetic database of over 60 V. fischeri genes and
ORFs that has given rise to a reliable codon-usage
table (E.G. Ruby, unpublished).
Recently developed molecular genetic tools have been
used to demonstrate that a number of determinants,
including motility (Graf et al., 1994), iron acquisition (Graf
and Ruby, 1994), amino acid prototrophy (Graf and Ruby,
1998), luminescence (Visick and Ruby, 1996), and a
periplasmic catalase (Visick and Ruby, 1998b), are required
by V. fischeri for normal symbiotic competence (Table 1).
Similarly, valuable new methods have been devised
for the study of the interaction of bacteria with specific E.
scolopes tissues and cell types, including:
(i) Development of a procedure by which to collect the
contents of the light organ crypts (Graf and Ruby, 1996;
Nyholm and McFall-Ngai, 1998),
(ii) Description of bacteria-induced changes in host cell
morphology (Foster and McFall-Ngai, 1998; McFallNgai, 1998b),
(iii) Identification of molecular markers for studies of
population genetics (Nishiguchi et al., 1998), and
(iv) Construction of green-fluorescent-protein (GFP)
expression vectors to label and visualize (by confocal
laser microscopy) V. fischeri and other Vibrio species
in the living host (McFall-Ngai, 1998b).
Taken together, these tools and approaches have put the
investigation of the squid-vibrio association in a position to
make significant advances at levels ranging from population
biology and ecology to cell biochemistry and molecular
genetics.
The Biology of the Association
The juvenile squid emerges from its egg free of bacteria
(i.e., ‘aposymbiotic’), and must obtain an inoculum of V.
fischeri cells to colonize its rudimentary light organ, and
thereby become bioluminescent (Wei and Young, 1989;
Montgomery and McFall-Ngai, 1994). In nature, inoculation
is achieved by the symbiosis-competent V. fischeri cells
typically present in the ambient seawater (Lee and Ruby,
1994a; 1995). The beating of cilia that cover two pairs of
lateral projections on the surface of the nascent light organ
(McFall-Ngai and Ruby, 1991; Montgomery and McFallNgai, 1993) serves to bring bacteria in the seawater close
to a series of external pores. These pores lead via ducts to
the interior of three pairs of crypts inside the light organ.
Within 12 hours a few V. fischeri cells have entered the
crypts, and have proliferated to a population size of
between 105 and 106 cells (Ruby and Asato, 1993).
As it grows and matures, the squid nourishes and
maintains within the light organ a V. fischeri population that
eventually reaches as many as 10 9 cells. This
nonpathogenic infection is characterized by two remarkable
traits: the symbiont population remains monospecific,
resisting any apparent contamination from other bacteria
in the external environment (Montgomery and McFall-Ngai,
1993); and, there is no opportunistic invasion of other host
Table 1. Examples of Colonization Determinants that Characterize Both Pathogenic and Benign Infections
Pathogenic Vibrio speciesa
Proposed Role
Colonization regulation
Secreted ADPr enzyme
Mucus penetration
Prototrophic growth
Iron acquisition
Adhesion
Catalase
Luminescence
Mannose-sensitive adhesion
Protease
hap regulation
a
Determinant
ToxR c
CtxAB c
motility f
HemA h
IrgA j
OmpU l
——
LuxAB o
MshA q
Hap s
HapR t
Colonization
phenotypeb
+
+
+
+
+
?
?
?
?
Non-pathogenic V. fischeri
Determinant
ToxR d
HvnA/B e
motility g
LysA i
GlnD k
OmpV m
KatA n
LuxAB p
(MshA) r
(Hap)
HapR u
Colonization
phenotype
?
?
+
+
+
+
+
+
+
?
?
This list is a compendium of characteristics attributed to V. cholerae, V. vulnificus, and/or V.
parahaemolyticus.
The absence of the determinant either is well correlated to a colonization defect (+), is not correlated to a defect (-), or is
as yet of an unclear or controversial importance (?).
c Salyers and Whitt, 1994
d Reich and Schoolnik, 1994
e
Reich and Schoolnik, 1996; Reich et al., 1997
f Richardson, 1991
g Graf et al., 1994
h
Rijpkema et al., 1992
i Graf and Ruby, 1998
j Tashima et al., 1996
k
Graf and Ruby, 1994; 1996
l Sperandio et al., 1995
m Aeckersberg and Ruby, 1998
n
Visick and Ruby, 1998b
o Palmer and Colwell, 1991
p Visick and Ruby, 1996; Visick et al., 1996
q
Jouravleva et al., 1998
r McFall-Ngai et al., 1998; Feliciano and Ruby, 1999
s Hase and Finklestein, 1993; Wu et al., 1996
t
Jobling and Holmes, 1997
u P.M. Fidopiastis and E.G. Ruby, unpublished
b
16 Ruby
tissues by these V. fischeri cells under normal
circumstances (Ruby and McFall-Ngai, 1992; Ruby, 1996).
These findings indicate the presence of a specific, tightly
regulated recognition between the bacteria and the tissues
of their host. In addition, population genetics studies (Lee
and Ruby, 1994b) have shown that, while strains of V.
fischeri isolated from different Euprymna species are
capable of initiating and sustaining a symbiosis with E.
scolopes, in mixed inoculations they are outcompeted by
native symbiont strains (Nishiguchi et al., 1998). Thus, the
squid-vibrio association is both a complex and an ancient
one, resulting from a coordinated evolution between the
two partners.
Ultrastructural studies of both juvenile and adult light
organs (McFall-Ngai and Montgomery, 1990; Montgomery
and McFall-Ngai, 1993; Nyholm and McFall-Ngai, 1998)
have revealed that V. fischeri associates with three types
of host cells within the crypts - the highly polarized
epithelium and specialized goblet cells that line the crypt
space, and a population of amoeboid granulocytes
(phagocytes) that moves freely within this space (Figure
2). These cell types all appear to participate in the dynamics
of the colonization process. The V. fischeri cells that initiate
the colonization must make their way through the ducts
leading to the light organ interior, adhere to the epithelium
of the crypts, and grow to fill the crypt spaces, utilizing
host-secreted organic material. Every day at dawn, the
squid expels most of the crypt contents, including 90 to
95% of the bacterial population, through the pores and into
the external environment (Ruby and Asato, 1993; Lee and
Ruby, 1994a); the remaining 5 to 10% of the bacteria
continue to be associated with the microvillous epithelium.
During the course of the day, these residual V. fischeri cells
proliferate, repopulating the crypts and restoring a full
bioluminescence potential for the host’s nocturnal behavior
(Boettcher et al., 1996). This continuous cycle of purging
and regrowth selects bacterial strains that not only are
capable of continued growth within the crypts, but also are
competitively dominant under the particular conditions created there (Lee and Ruby, 1994b; Visick and Ruby, 1998b).
Because the pores leading to the crypts remain open
to the environment throughout the life of the squid,
inappropriate species of bacteria that are present in the
ambient seawater will continue to have access to the crypts
(Lee and Ruby, 1994a). One line of defense against
colonization by bacteria other than symbiosis-competent
V. fischeri may be the phagocytic cells that are present in
the crypts (Nyholm and McFall-Ngai, 1998). The function
of these phagocytes is not well defined in the squid-vibrio
system, but they are members of the class of cells that are
the principal mediators of the immune response in squids
(Budelmann et al., 1997). These cells are thus candidates
for a role in maintaining the light organ species-specific for
V. fischeri, and/or in controlling the size of the symbiont
population.
Cellular Events Occurring During Colonization
Detailed morphological, biochemical and genetic studies
have revealed a number of dramatic events that
accompany the initiation and persistence of the benign
infection of the light organ crypts by V. fischeri cells. During
the first few hours to days following the colonization of the
crypt epithelium, both partners undergo significant
symbiosis-related changes. For instance, the bacteria
differentiate into a non-flagellated form (Ruby and Asato,
1993), and their level of luminescence per cell is induced
over 1000-fold (Boettcher and Ruby, 1990) due to the
accumulation in the crypts of at least one V. fischeri-specific
quorum-sensing molecule, an acyl-homoserine lactone
called VAI-1 (Fuqua et al., 1996; Boettcher and Ruby,
1995).
The squid host undergoes an even more striking
developmental program. Morphologically, before
colonization is initiated, the nascent light organ has two
pairs of ciliated projections on its surface (Montgomery and
McFall-Ngai, 1993), and the polarized epithelial cells that
line the crypts are columnar and have sparse microvillar
projections (Lamarcq and McFall-Ngai, 1998). After V.
fischeri cells have initiated their cooperative interaction,
the gross morphology of these light organ structures begins
to change. Within 8 hours following inoculation the ciliated
projections begin to regress through a process of
b
b
v
b
A
B
C
Figure 2. Transmission Electron Micrographic Images of Host Cell Types Encountered in the Light Organ by Symbiotic Vibrio fischeri
(A) Epithelial cells lining the light organ crypts bear dense microvilli in which V. fischeri cells (b) are embedded (bar = 2 µm). (B) Scattered throughout the
epithelia, and near the bacteria, are goblet cells that contain a high density of internal vesicles (v), possibly containing glycoproteins (bar = 5 µm). (C)
Phagocytic mollusk granulocytes are found within the crypts, moving among the bacterial population (bar = 1 µm).
The Squid-Vibrio Symbiosis as a Biomedical Model 17
programmed cell death (Montgomery and McFall-Ngai,
1994; Foster and McFall-Ngai, 1998), and after a few days
they have completely disappeared (Doino and McFall-Ngai,
1995). In addition, the epithelial cells lining the crypts have
increased 4-fold in volume and become more cuboidal,
and their microvilli have increased 4-fold in density and
more completely surround and support the bacterial cells
(Figure 2A) (McFall-Ngai, 1994; Lamarcq and McFall-Ngai,
1998). There are also significant biochemical changes
during this period. Initially, a high concentration of a species
of host mRNA encoding haloperoxidase (HPO) (Weis et
al., 1996) is present in the uncolonized light organ.
However, once the organ is colonized, the level of HPO
drops significantly (Small and McFall-Ngai, 1998), thus
reducing the potential for the production of the antibacterial
compound hypohalous acid (Forman and Thomas, 1986;
Tomarev et al ., 1993; Boettcher, 1994). The host
developmental program is not initiated in the absence of a
symbiotic infection (McFall-Ngai and Ruby, 1991); however,
once triggered, some (but not all) of the events will proceed
without the continued presence of the bacteria (Doino and
McFall-Ngai, 1995; Lamarcq and McFall-Ngai, 1998). As
a result, a distinct, functional organ is formed through a
program of biochemical accommodation and cooperation
that is analogous to the well described development of the
Rhizobium -legume symbiosis (van Rhijn and
Vanderleyden, 1995).
Obstacles to Bacterial Colonization
To colonize an animal host, bacteria, whether pathogenic
or cooperative, must overcome characteristics of the tissue
that discourage the growth of inappropriate
microorganisms. These obstacles to colonization may
include general environmental conditions in the tissue, as
well as active responses by the host’s defenses. Five such
challenges that appear to play a role in colonization of the
light organ crypts are summarized below.
Viscous Barrier
Both flagellated and unflagellated motility mutants of V.
fischeri are unable to initiate a colonization, even when
presented as a 1000-fold higher inoculum, and for a 10times longer exposure (Graf et al., 1994). The cilia lining
the duct leading to the crypts create an outward-directed
current (McFall-Ngai and Ruby, 1998), and the presence
of mucopolysaccharides in the ducts (Nyholm and McFallNgai, 1998) and emanating from the pore (Figure 3)
suggests that the cells must penetrate a viscous barrier to
reach the crypts themselves.
m
Figure 3. Mucus-like Material Around the Pore Leading to the Light Organ Crypts of Euprymna scolopes
Transmission electron micrographs reveal a flocculant, glycoprotein-like material (m) surrounding the ciliated cells that form the pore (arrow) leading into the
crypt spaces (bar = 1 µm).
18 Ruby
Nutritional Limitation
Proteins and/or peptides in the crypt contents may
constitute a substantial source of nutrients: the ability of
some amino acid auxotrophs to fully colonize the crypts
indicates that the host must be a major source of these
building blocks (Graf and Ruby, 1998). While it is not yet
known whether there is a specific bacterial signal that is
needed to elicit the host’s production and export of these
nutrients, two morphological observations of the colonized
light organ are suggestive. First, the presence of goblet
cells has been noted in both the duct and crypt regions of
the light organ (Figure 2B). These goblet cells could be
the source of mucopolysaccharide glycoproteins, like
mucin, that might be secreted into the crypts. Second, the
epithelial cells lining the crypt walls become edematous,
swelling to about 4-fold the volume of the cells lining
uninfected crypts (Montgomery and McFall-Ngai, 1994).
Such swelling is indicative of osmotic stress in epithelial
cells, which may lead to the leaking of organic materials
into the surrounding extracellular spaces.
Clearing by Expulsion
The diurnal process of expulsion will select for symbionts
that are able to attach to the epithelial cells lining the crypt
spaces. Evidence exists to suggest that a mannosesensitive adhesion is important for successful colonization
(McFall-Ngai et al., 1998; Feliciano and Ruby, 1999). In
addition, the increasing intimacy observed between V.
fischeri cells and the epithelial crypt microvilli as the
association matures (Lamarcq and McFall-Ngai, 1998)
suggests that bacterial outer membrane proteins
(Aeckersberg and Ruby, 1998) or other surface
components become increasingly important for persistent
attachment.
Exposure to Reactive Oxygen Species
Recent data indicate that the uninfected crypt environment
is oxidatively stressful; for instance, elevated superoxide
anion production has been detected in the light organ tissue
(Small and McFall-Ngai, 1993). In addition, the squid light
organ has unusually high levels of mRNA encoding a HPO
with significant sequence similarity to human
myeloperoxidase, an antimicrobial protein in neutrophils
(Tomarev et al., 1993) that catalyzes the conversion of H2O2
and halide ions into microbicidal hypohalous acids. Activity
assays and immunocytochemical analyses have
demonstrated that HPO is present in the crypt space, and
occurs at elevated levels elsewhere in the squid where
there are pathogenic bacterial infections (Small and McFallNgai, 1998).
Removal by Phagocytes
One line of defense against colonization that V. fischeri
cells must avoid appears to be the phagocytic cells in the
crypts (Figure 2C) (Nyholm and McFall-Ngai, 1998). Such
cells arise in a tissue called the white body and are typically
found in the hemolymph (Budelmann et al., 1997) and in
extracellular spaces like the light organ crypts. Phagocytes
of invertebrates share several ultrastructural, physiological
and biochemical traits with mammalian cells such as
neutrophils and macrophages that are associated with
innate immunity (Sminia et al., 1987; Greger et al., 1996).
These traits underlie the common responses of these cells
to microorganisms, such as respiratory burst activity and
phagocytosis. Such common response pathways, which
can function through the release of chemokines and
cytokines (e.g., interleukins and tumor necrosis factoralpha), are indicative of an ancient origin for the
mechanisms by which animal cells and microorganisms
interact (Belvin and Anderson, 1996; May and Ghosh,
1998). Continuing studies of these and other host-imposed
barriers are an important focus of research into the basis
of the reciprocal accommodation reached between V.
fischeri and its host during the development of the light
organ association.
Similarities between Benign and Pathogenic
Tissue Colonization
In recent years it has become clearer that certain virulence
determinants of pathogenic Vibrio species are expressed
in, and may be required for, the benign V. fischeri
colonization. The list of similarities between the colonization
of host epithelium by V. fischeri and that of a number of
pathogenic Vibrio species such as V. vulnificus , V.
parahaemolyticus and V. cholerae continues to grow (Table
1). These similarities include known virulence determinants
of V. cholerae (ToxR and CtxAB) that have protein
homologs or analogs in V. fischeri (Reich and Schoolnik,
1994; Reich and Schoolnik, 1996; Reich et al., 1997).
Because of these similarities, and the phylogenetic
relatedness of V. fischeri and pathogenic Vibrio species, it
is reasonable to hypothesize that a number of properties
of their associations with host tissue might have arisen
from a common evolutionary origin. In fact, some species,
like V. vulnificus and V. harveyi, can be a benign symbiont
in one host and a virulent pathogen in another (DePaola
et al., 1994; Harris et al., 1996) suggesting a duality of
function in these different classes of association. This
hypothesis is further supported by additional evidence of
the homologous character of tissue colonization by V.
fischeri and pathogenic Vibrio species. The identity of these
V. fischeri genetic and enzymatic homologs suggests that
they are important in three critical and interacting aspects
of epithelial colonization: bacterial attachment, bacterial
avoidance of host defense responses, and host provision
of nutrition to the bacterial culture (Table 1). Because the
commensal colonization of invertebrate tissues by Vibrio
species arose millions of years before the development of
human pathogenesis by members of this genus it seems
likely that host-colonization mechanisms that were present
in these cooperative associations may have been recruited
into the development of pathogenic associations. A similar
argument has been made to explain the existence of a
majority of the known Salmonella virulence determinants
in nonpathogenic Escherichia coli, the organism in which
many of these determinants were first discovered and
described (Groisman and Ochman, 1994)
The epithelial cell surfaces of the human body harbor
a complex and essential community of bacterial species.
These bacteria not only create a normal, healthy
biochemical microenvironment on these surfaces, but also
provide the first line of defense against invading pathogens.
The growing awareness of the essential role that the natural
microbiota play in the development and maintenance of
the healthy state in animals (Hooper et al., 1998) has led
to an increasing recognition of the importance of benign
bacterial colonization of tissues. To design effective
The Squid-Vibrio Symbiosis as a Biomedical Model 19
therapeutic measures against potential diseases, we must
understand the dynamic interactions between hosts and
their essential microbial partners. However, little is known
either about the signaling processes, and their genetic
regulation, through which these beneficial bacteria establish
stable associations with host cells, or about how these very
same mechanisms are exploited by harmful bacteria.
The light organ symbiosis between E. scolopes and
V. fischeri offers unique opportunities to study these
processes. Important characteristics of this natural
experimental model that make it particularly useful for
comparisons of benign and pathogenic colonization of
animal tissue are: (i) the laboratory culturability of both
partners (Wei and Young, 1989; Boettcher and Ruby, 1990;
Hanlon et al., 1997); (ii) the specificity of the association
(McFall-Ngai and Ruby, 1991); (iii) the similarity between
the microvillous, polarized epithelial cells and phagocytes
of the light organ crypts and those of the human intestinal
lining (McFall-Ngai and Montgomery, 1990; Nyholm and
McFall-Ngai, 1998); and, (iv) the close genetic relationship
between V. fischeri and pathogenic relatives, such as V.
vulnificus and V. cholerae.
As in most associations between bacteria and their
hosts, this benign infection does not produce a diseased
state in the colonized tissue; thus, the signals and
responses of the two partners follow a predictable and
integrated pattern. However, unlike the most common types
of associations (e.g., the normal microbiota of the enteric
tract, skin and oral cavity), the V. fischeri light organ
colonization is monospecific, thereby greatly simplifying
the elucidation of bacteria-host interactions. To date,
success with this model has resulted from collaborations
involving bacterial molecular physiologists and geneticists,
and animal developmental biochemists. By continuing to
integrate efforts to understand the responses of each
member of this association to the other, the chances of
making important discoveries about bacterial tissue
colonization will be enhanced.
Future Directions
Further investigations of the symbiotic colonization of E.
scolopes by V. fischeri will promote a paradigm for studying
host-bacterial interactions that will specifically serve to
advance biomedical objectives in the following ways.
(i) Defining the biochemical and molecular events that
can characterize the specific initiation, rapid
colonization, and long-term persistence of benign
bacterial infections is of increasing importance. While
a number of other promising model systems are
emerging (Forst and Nealson, 1996; Whittaker et al.,
1996; Hooper et al., 1998; Graf, 1999), the squid-vibrio
association is already becoming well-established
(Ruby, 1996; McFall-Ngai, 1998b).
(ii) The importance of the kind of bacterial signaling called
“quorum-sensing” (Fuqua et al., 1996) in pathogenic
bacterial-host signal interactions has become
increasing evident. V. fischeri, the species in which
this mechanism was first discovered, continues to be
the paradigm system for the study of both intra- and
interspecies association (Visick and Ruby, 1999).
(iii) Continued studies of bacterial and host determinants
that potentiate light organ symbiosis may not only
reveal further convergences with known pathogenic
virulence factors (Table 1) but, more importantly, may
also promote the discovery of as yet undescribed ones
(Fidopiastis and Ruby, 1999). In addition, the level of
understanding of the ecology of V. fischeri (Ruby and
Lee, 1998) makes this species particularly wellpositioned for use in testing experimental models of
the evolution of horizontally transmitted pathogens
(Ebert, 1998).
(iv) Future discoveries of the biology of the squid-vibrio
association may aid in efforts to understand the
mechanisms by which benign colonizations of mollusk
tissue serve as a reservoir for human pathogenic Vibrio
species (Tamplin and Capers, 1992), and to determine
the role of such reservoirs in the recent resurgence of
pathogenic epidemics (Colwell, 1996).
Acknowledgements
I thank M. McFall-Ngai and her laboratory for their critical collaborative
interactions, and specifically for all the images used in this review. I also
thank the members of my laboratory for producing much of the data I’ve
discussed.
Portions of the research described herein were funded by the National
Institutes of Health through grant RO1-RR12294 to E.G.R. and M. McFallNgai, and by National Science Foundation grant IBN96-01155 to M. McFallNgai and E.G.R.
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